Abstract
Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMP-dependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC50 ∼10 nM, maximum at 1 μM) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of <10 μM AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC50 ∼50 nM and ∼16 μM, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of ≤10 μM AS in the presence of SOD but caused NO release from 0.1 mM AS in the absence of SOD. These data indicate that AS activates sGC exclusively via NO, formed either via SOD-catalyzed oxidation of HNO or through a minor AS decomposition pathway that is unmasked in the presence of HNO scavenging thiols.
- sGC, soluble guanylate cyclase
- AS, Angeli's salt (Na2N2O3, sodium trioxodinitrate)
- carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide
- DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NONOate)
- DTPA, diethylene triamine pentaacetic acid
- DTT, dithiothreitol
- ODQ, 1H-(1,2,4)oxadiazolo(4,3-α) quinoxaline-1-one
- SIN-1, 3-morpholino sydnonimine
- SOD, superoxide dismutase
- TEA, triethanolamine
- HNO, nitroxyl.
Footnotes
-
↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
-
This work was supported by the Fonds zur Föderung der Wissenschaftlichen Forschung in Austria [Grants W901 Molecular Enzymology and P20669] and the Deutsche Forschungsgemeinschaft [Grant KO1157/4-1].
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.059915
-
ABBREVIATIONS:
- Received July 30, 2009.
- Accepted August 31, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|